Engineering Hetero-Bivalent Ligands to Inhibit IgE Clustering on Mast Cells Project Summary/Abstract Allergy is a disorder of the immune system, which is elicited by the allergen-mediated-clustering of the immunoglobulin E (IgE) molecules on the surface of mast cells and basophils. It is estimated that up to 12 million Americans have food allergies, 10 million suffer from allergic asthma, 10 million suffer atopic eczema, 36 million have allergic rhinitis, and the prevalence is rising. The majority of the current therapies used to treat allergy related adverse immunologic responses involve non-specific suppression of the immune system. This results in the increased risk of infectious diseases, highlighting the need to develop selectively targeting treatment alternatives. Multivalent binding interactions provide higher affinity (termed avidity), and result in stronger interactions. Earlier studies have established that bivalent binding, as in the case of antibodies, can strengthen the avidity of an interaction up by six orders of magnitude over monovalent binding. Our overall goal in this proposed project is to engineer hetero-bivalent ligands that will competitively and selectively inhibit allergen binding to IgEs on mast cells, thereby inhibit IgE clustering and allergic responses. This will be accomplished via designing hetero-bivalent molecules that simultaneously target two nearby binding sites located on the Fab domain of an IgE: the antigen binding site; and the not-so-well-known nucleotide binding site. Simultaneous bivalent binding to both these sites will provide the hetero-bivalent ligand with enhanced avidity and selectivity for the target IgE, and will enable competitive inhibition of allergen binding to the IgEs. These hetero-bivalent ligands will be designed to have two moieties - the antigenic molecule itself (or the mimotope), and the nucleotide binding site-targeting molecule - covalently linked by a flexible linker molecule. Specifically we will: i) identify nucleotide analogs that will target the unconventional nucleotide binding site on the IgE; ii) design and synthesize hetero-bivalent ligands of 'hapten/nucleotide-analog' conjugates; iii) determination of the Kd for hetero-bivalent ligand - IgE interactions and evaluation of their inhibitory effect on the allergen binding to IgEs and iv) determine the efficacy of the hetero-bivalent ligands to inhibit allergic responses in vitr cellular assays.